Compositions and methods for generating a persisting population of T cells useful for the treatment of cancer

ABSTRACT

The present invention provides compositions and methods for generating a genetically modified T cells comprising a chimeric antigen receptor (CAR) having an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain, wherein the T cell exhibits prolonged exponential expansion in culture that is ligand independent and independent of the addition of exogenous cytokines or feeder cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2013/027337, filed Feb. 22, 2013, which claims priority to U.S.Provisional Application No. 61/601,890, filed Feb. 22, 2012, all ofwhich applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant 1R01CA120409awarded by the National Cancer Institute. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The generation of tumor-specific T lymphocytes by genetic modificationto express chimeric antigen receptors (CARs) is gaining traction as aform of synthetic biology generating powerful antitumor effects (Jena etal., 2010, Blood. 116:1035-1044; Bonini et al., 2011, Biol Blood MarrowTransplant 17(1 Suppl):515-20; Restifo et al., 2012, Nat Rev Immunol12:269-281; Kohn et al., 2011, Mol Ther 19:432-438; Savoldo et al.,2011, J Clin Invest 121:1822-1825; Ertl et al., 2011, Cancer Res71:3175-3181). Because the specificity is conferred by antibodyfragments, the CAR T cells are not MHC restricted and are therefore morepractical than approaches based on T cell receptors that require MHCmatching.

Clinical data from patients treated with CD19-specific CAR⁺ T cellsindicates that robust in vivo proliferation of the infused T cells is akey requirement for immunoablation of tumors (Porter et al., 2011, NEngl J Med 365:725-733; Kalos et al., 2011, Sci Transl Med 3:95ra73).Therefore, efforts have been made to incorporate the signalingendodomains of co-stimulatory molecules such as CD28, OX40, and 4-1BBinto CARs. In 1998 it was first reported that the use of gene-engineeredT cells expressing chimeric single-chain (scFv) receptors capable ofco-delivering CD28 costimulation and T cell receptor/CD3 zeta chain(CD3ζ) activation signals increased the function and proliferation ofCAR T cells (Krause et al, 1998, J Exp Med 188:619-626; Finney et al.,1998, Journal of Immunology 161:2791-2797). A number of laboratorieshave confirmed that incorporation of CD28 signaling domains enhances thefunction of CARs in pre-clinical studies compared to CD3ζ or FcεR1(Geiger et al., 2001, Blood 98:2364-2371; Arakawa et al., 2002,Anticancer Research 4285-4289; Haynes et al., 2002, J Immunol169(10):5780-6; Maher et al., 2002, Nature Biotechnology 20:70-75;Finney et al, 2004, J Immunol 172:104-113; Gyobu et al., 2004, CancerRes 64:1490-1495; Moeller et al., 2004, Cancer Gene Ther 11:371-379;Teng et al., 2004, Hum Gene Ther 15:699-708; Friedmann-Morvinski et al.,2005, Blood 105:3087-3093; Pule et al., 2005, Molecular Therapy12:933-941; Westwood et al., 2005, Proc Natl Acad Sci USA102:19051-19056; Willemsen et al., 2005, J Immunol 174:7853-7858;Kowolik et al, 2006, Cancer Res 66:10995-11004; Loskog et al., 2006,Leukemia 20:1819-1828; Shibaguchi et al., 2006, Anticancer Res26:4067-4072; Brentjens et al., 2007, Clin Cancer Res 13:5426-5435; Tenget al., 2006, Human Gene Therapy 17:1134-1143). In a study in patientswith B-cell malignancies, CD28:CD3ζ CARs had improved survival comparedto CARs endowed only with the CD3ζ signaling domain (Savoldo et al.,2011, J Clin Invest 121:1822-1825).

However, there is still a need in the art to better improve constructionof CARs that permit extensive T-cell proliferation. The presentinvention satisfies this need in the art.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen binding domain, a hinge domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, andfurther wherein when the CAR is transduced into a T cell, the CARcontributes to at least one of: increased antigen-independent activationof the transduced T cell, increased mean cell volume (MCV) of thetransduced T cell, increased cell population expansion of the transducedT cell, increased proliferation of the transduced T cell, increasednumbers of progeny of the transduced T cell, increased effector cytokinesecretion, sustained expression of granzyme, increased persistence ofthe transduced T cell population in vitro, or increased persistence ofthe transduced T cell population in vivo.

In one embodiment, the hinge domain is an IgG4 hinge domain.

In one embodiment, the antigen binding domain is an anti-cMet bindingdomain, the hinge domain is IgG4, the transmembrane domain is a CD28transmembrane domain, and the costimulatory signaling region is a CD28signaling region. In one embodiment, the CAR comprises the amino acidsequence of SEQ ID NO: 1.

In one embodiment, the antigen binding domain is an anti-mesothelinbinding domain, the hinge domain is an IgG4 hinge domain, thetransmembrane domain is a CD28 transmembrane domain, and thecostimulatory signaling region is a CD28 signaling region. In oneembodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the antigen binding domain is an anti-CD19 bindingdomain, the hinge domain is an IgG4 hinge domain, the transmembranedomain is an CD28 transmembrane domain, and the costimulatory signalingregion is a CD28 signaling region. In one embodiment, the CAR comprisesthe amino acid sequence of SEQ ID NO: 3.

In one embodiment, the antigen binding domain is an antibody or anantigen-binding fragment thereof.

The invention also provides a T cell comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the CAR comprising anantigen binding domain, a hinge domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, andwherein when the CAR is transduced into a T cell, the CAR contributes toat least one of: increased antigen-independent activation of thetransduced T cell, increased mean cell volume (MCV) of the transduced Tcell, increased cell population expansion of the transduced T cell,increased proliferation of the transduced T cell, increased effectorcytokine secretion, increased expression of granzyme, increased numbersof progeny of the transduced T cell, increased persistence of thetransduced T cell population in vitro, or increased persistence of thetransduced T cell population in vivo.

The invention also provides a vector comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the CAR comprising anantigen binding domain, a hinge domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, andwherein when the CAR is transduced into a T cell, the CAR contributes toat least one of: increased antigen-independent activation of thetransduced T cell, increased mean cell volume (MCV) of the transduced Tcell, increased cell population expansion of the transduced T cell,increased proliferation of the transduced T cell, increased numbers ofprogeny of the transduced T cell, increased persistence of thetransduced T cell population in vitro, or increased persistence of thetransduced T cell population in vivo.

The invention also provides a persisting population of geneticallymodified T cells, wherein the T cells comprise a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the CAR comprising anantigen binding domain, a hinge domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, andwherein when the CAR is transduced into a T cell, the CAR contributes toat least one of: increased antigen-independent activation of thetransduced T cell, increased mean cell volume (MCV) of the transduced Tcell, increased cell population expansion of the transduced T cell,increased proliferation of the transduced T cell, increased numbers ofprogeny of the transduced T cell, increased persistence of thetransduced T cell population in vitro, or increased persistence of thetransduced T cell population in vivo.

In one embodiment, the genetically modified T cells exhibit ananti-tumor immunity when the antigen binding domain binds to itscorresponding antigen.

In one embodiment, the persisting population of genetically modified Tcells of exhibit a cytokine signature comprising at least one cytokineselected from the group consisting of IFN-γ, TNF-α, IL-17A, IL-2, IL-3,IL-4, GM-CSF, IL-10, IL-13, Granzyme B, Perforin, and any combinationthereof.

In one embodiment, the T cells proliferate in the absence of exogenouscytokine or feeder cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A through 1B, is a series of images depictingchimeric antigen receptor constructs and relative expression levels.FIG. 1A shows a representation of CAR constructs depicting the variousscFv, hinge regions, transmembrane and cytosolic domains. All CARscontain the CD28 and CD3ζ intracellular signaling domain except for theCART19 CAR which contains the 4-1BB rather than CD28 intracellulardomain. FIG. 1B demonstrates that the surface expression of each CARconstruct in (FIG. 1A) was analyzed 6 days following lentiviraltransduction to quantify relative expression levels.

FIG. 2, comprising FIGS. 2A through 2D, is a series of images depictinginduction of constitutive, ligand independent CD4 CAR T cellproliferation. FIG. 2A shows the in vitro proliferation of human CD4+ Tcells following 5 days of αCD3/CD28 coated magnetic bead stimulation andlentiviral transduction with the indicated CAR constructs (left panel).The c-Met IgG4 CAR and both SS1 CARs exhibit constitutive proliferationfor 60 days. No cytokines were added to culture media at any pointduring expansion. The CAR T cells with constitutive proliferation alsomaintain a larger mean cell volume (right panel). Results arerepresentative of n>10 normal human donors. FIGS. 2B and 2C demonstratethat CD4+ and CD8+ T cells were stimulated as in (FIG. 2A), with orwithout exogenous IL-2. FIG. 2D demonstrates that CD4+ T cells from 3healthy donors were isolated, stimulated and transduced with lentivirusencoding the c-Met IgG4, CD19 CD8-α, and CART19 CAR constructs or mocktransduced, and cultured with addition of fresh media and no exogenouscytokines. Error bars denote standard deviation.

FIG. 3, comprising FIGS. 3A and 3B, is a series of images demonstratingthat CAR T cells with continuous T cell proliferation have constitutivecytokine secretion. FIG. 3A depicts serial measurements of cytokineproduction by various CAR constructs following αCD3/CD28 stimulation andexpansion. At each noted time point c-Met IgG4, CD19 CD8-α CARtransduced, and untransduced CD4+ T cells were collected from culture,washed and re-plated at 1×10⁶/mL. Cells were kept in culture for 24 hrsat which time supernatant from each culture was collected. Supernatantswere analyzed via luminex assay and values plotted as log(10) foldchange from the pre-stimulated cells (baseline). Baseline values (pg/ml)for each analyte were: IFN-γ: 3.66 pg/mL; TNF-α: 0.29 pg/mL; IL-2: 0.51pg/mL; GM-CSF: 4.58 pg/mL; IL13: 4.79 pg/mL; IL-10: 1.29 pg/mL. FIG. 3Bshow supernatant from CARs displaying the growth phenotype inducesactivation of naïve unstimulated T cells. Culture supernatant from c-MetIgG4 CAR T cell culture harvested on day 56 of culture was added tounstimulated naïve CD4+ T cells at a final concentration of 12.5%, 25%,or 50% c-Met IgG4 supernatant relative to starting media. As controls,media with and without 100 IU of IL-2 were also included, as well asCD3/CD28 bead stimulated cells kept in culture with initial stimulationon day 0 and re-stimulation on day 12. MCVs were determined and cellmedia was added every two days to maintain the supernatant concentrationand IL-2 concentration within control group as described elsewhereherein.

FIG. 4, comprising FIGS. 4A and 4B, is a series of images demonstratingthat CARs with a constitutive growth phenotype display a unique genesignature. FIG. 4A depict cytokines, perforin and granzyme expression.Microarray analysis comparing cytokine expression of c-Met IgG4, CD19CD8-α, CART19 CARs and untransduced T cells at baseline and on days 6,22 and 24 of culture; only the c-Met IgG4 culture was analyzed on day 24because the other cultures were terminated due to cell death. Noexogenous cytokines were added to the culture media. Normalized absolutelog₂ gene expression intensities are plotted for IFN-γ, TNF-α, IL-17A,IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, Granzyme B and Perforin, FIG. 4Bdemonstrates that CAR T cells with a constitutive growth phenotypedisplay distinct transcription factors. Expression of genes importantfor T cell polarization, growth and survival: T-bet, Eomes, GATA-3,RORc, FoxP3, Bcl-xL, KLRG1, and hTERT. Normalized absolute log(2) geneexpression intensities are plotted. Data is compilation of normal donortriplicates analyzed prior to stimulation, and on days 6, 11 and 24;only the c-Met IgG4 culture is analyzed on day 24 because the othercultures were terminated due to cell death. Each dot denotes a singledonor within each time point expressing either the c-Met IgG4 CAR, CD19CD8α CAR, or untransduced control. Box plots representing upper 75^(th)and lower 25^(th) percentile with median. Whiskers denote upper 90^(th)and lower 10^(th) percentile. Comparison of c-Met IgG4 CAR vs CD19 CD8αCAR (red) on day 11 by ANOVA: T-bet (p=0.888); Eomes (p=0.003); GATA-3(p<0.001); FoxP3 (p=0.122); RORc (p=0.089); KLRG1 (P=0.076); hTERT(p=0.405); and Bcl-xL (p<0.001).

FIG. 5, comprising FIGS. 5A and 5B, is a series of images demonstratingthat constitutive activation of AKT, NF-kB and MAPK signaling pathwaysis associated with the CAR T cell proliferative phenotype. FIG. 5A showsa representative FACS histograms displaying enrichment of c-Met IgG4 CAR(+) T cells during culture from day 10 to day 30 of culture. FIG. 5Bdepicts PhosFlow plots of CD4+ T cells stimulated and transduced withthe c-Met IgG4 or CD19 CD8αCARs. On days 6, 10 and 25 cells were fixed,permeabilized and stained using PE anti-Erk1/2 (pT202/pY204), PEanti-Akt (pS473), PE anti-NF-kB p65 (pS529) and PE anti-S6(pS235/pS236); the CD19 CD8α CAR culture did not continue to proliferateto day 25, and therefore is only analyzed on days 6 and 10. Positivecontrols were samples from each condition stimulated for 10 min usingPMA/Ionomycin prior to fixation, while negative controls cells werefully stimulated T cells stained using PE conjugated IgG2b κ isotypecontrol.

FIG. 6, comprising FIGS. 6A through 6C, is a series of imagessummarizing results from a genome-wide microarray analysis of CAR Tcells with constitutive proliferation. FIG. 6A demonstrates that CD4+ Tcells from 3 donors expressing continuous c-Met IgG4 or classic CD19CD8α CARs, or mock transduced cells were subjected to microarrayanalysis and hierarchical clustering from day 0 to day 24 of culture.Clustering was done using the euclidean distance of median normalizedabsolute log(2) gene expression intensities with average linkage. Theplots are based on unbiased whole genome clustering. On day 11, CD19CD8α CART cells and untransduced cells cluster more similarly to restingT cells, while day 11 and day 24 c-Met IgG4 CAR T cells remain activatedand closely cluster. FIG. 6B shows a distinct gene expression signatureof CAR T cells with constitutive proliferation. The gene expressionsignatures from the 3 donor T cell cultures on day 6 is compared to day11 and day 24 cultures. CAR T cells on day 6 are similar to mocktransduced T cells. In contrast, on day 11 and day 24 the continuousc-Met IgG4 cells display a unique RNA signature that differs from thefully activated day 6 phenotype. FIG. 6C shows the differences inexpression of genes between c-Met CAR and CD19 CAR.

FIG. 7, comprising FIGS. 7A through 7C, is a series of imagesdemonstrating that transgene expression levels are sufficient to conveythe constitutive CAR growth phenotype. In vitro proliferation of humanCD4+ T cells following 5 days of anti-CD3 plus CD28 stimulation andlentiviral transduction with c-MET expressing CARs under the indicatedpromoter. CMV(1) and CMV(2) represent replications of lentiviral vectorproduction in the same human donor. FIG. 7A demonstrates that populationdoublings were determined for both CMV and EF-1α driven c-MET CAR cells.After ˜12 days in culture, CMV-c-MET CAR cells were unable to sustainproliferation and died, while EF-1α c-MET CAR T cell continue toproliferate. FIG. 7B demonstrates that Mean cell volume (MCV) was alsodetermined. The CMV-c-MET CAR T cells decreased in cell size after 10days, indicative of the cells resting down. FIG. 7C depicts a comparisonof the level of expression between CARs expressed with the CMV and EF-1αpromoters at day 6 post-transduction. The mean fluorescence intensity isindicated.

FIG. 8 is an image showing CAR T cells with constitutive proliferationretain specific cytotoxicity. The M30 tumor line (endogenous expressionof c-Met), and the NCI-H522 tumor line (lacking c-Met expression) werecultured at the indicated effector to target ratio with c-Met IgG4 CARTcells. CD19 CD8α CAR T cells were used as specificity controls toexclude allogeneic effects. Inset boxes: c-Met expression on M108 andNCI-H522.

FIG. 9 is an image demonstrating that c-Met and mesothelin expression isnot detected on human CD4+ T cells. CD4+ T cells do not expressdetectable levels of c-Met or mesothelin. Samples were compared to L55,a non-small cell lung tumor cell line, as well as unstained CD4+ Tcells. Following activation, human CD4+ T cells and tumor lines werestained for c-Met (PE) or mesothelin (PE). Histograms depict unstainedactivated CD4+ T cells, activated CD4+ T cells, and the L55 tumorstained for described antigen. The mean fluorescence intensity isindicated.

FIG. 10 is an image of a Western blot analysis of CARs with constitutiveor inducible proliferative phenotype. Western blot was performed underreducing and non-reducing conditions probing for CD3ζ using mouseanti-human CD3ζ at 0.250 ug/mL followed by anti-mouse HRP at 1:5000 onlysates from day 8 post transduction samples. CAR monomers (50 to 70 kD)under reducing conditions (left) and dimers and monomers undernon-reducing conditions (right). Endogenous CD3ζ as internal loadingcontrol.

FIG. 11 is an image demonstrating that CAR T cells with a constitutivegrowth phenotype retain a diverse TCR Vβ repertoire. Human CD4 T cellswere isolated, stimulated with anti-CD3/CD28, transduced with c-Met IgG4CAR, and maintained in culture without exogenous cytokines as described.Donor matched mock transduced cells were stimulated and expandedsimultaneously as control, however these cultures required additionalstimulations to maintain in culture. Cells were cryopreserved at days 0,13 and 34 after which they were simultaneously thawed and TCR Vβanalysis was performed using the IOTest Beta Mark TCR V kit.

FIG. 12 is an image demonstrating that CAR T cells with constitutiveproliferation have ligand-independent NFAT activation. Jurkat T cellsengineered to express GFP under the control of the NFAT promoter weretransduced with lentivirus encoding CARs for continuous c-Met IgG4, SS1IgG4, SS1 CD8α, and classic CARs encoding CD19 IgG4, CD19 CD8α, and SS1CD8aΔtail. Cells were analyzed 3 days following transduction for GFP andCAR expression

FIG. 13 is an image demonstrating that constitutive CAR T cellproliferation results in differentiation and evolution of a distinctcell surface phenotype. CD4 T cells were stimulated and transduced withthe c-Met IgG4 CAR construct as previously described. Pre-stimulationcells were cryopreserved for later analysis. Cell samples were isolatedat day 6, 14, 23, 38 and 70 and cryopreserved. Cells were thawedsimultaneously and allowed to rest overnight without addition ofcytokines. Cells were stained for CAR as well as CD25, CD70, PD-1, CD27,CD28, CD62L, CCR7 and Crtam.

FIG. 14 is an image depicting effects of stimulation and cell culture ondifferentiation of non-transduced T cells. Companion CD4 T cells wereisolated via negative depletion and stimulated concurrently with the CART cells shown in FIG. 12. Pre-stimulation cells were cryopreserved forlater analysis. Cell samples were isolated for analysis at day 6, 24hours following bead removal, and on day 14. Cells were thawedsimultaneously with the CAR T cells and rested overnight withoutadditional of growth factors or cytokines. Cells were analyzed for CARexpression as well as CD25, CD70, PD-1, CD27, CD28, CD62L, CCR7 andCrtam.

FIG. 15 is an image depicting temporal patterns of telomere restrictionfragment length (TRF) in continuous CAR T cells and mock transduced Tcells. CD4 T cells transduced with the continuous c-Met IgG4 CAR or mocktransduced cells were cultured for the indicated duration. DNA wasisolated from the T cells and terminal telomeric restriction fragmentlength assessed by electrophoretic separation of HinfI/RsaI digested DNAfollowed by in-gel hybridization to a telomere repeat probe. Thecontinuous CAR T cells proliferated for at least 61 days in culturewhile the mock transduced T cells ceased proliferation after 31 days.The ladder is 32P-labeled mixture of full-length and HindIII-digestedlambda DNA.

FIG. 16 is an image depicting engraftment and proliferation ofcontinuous CAR T cells in NSG mice. Human CD4 T cells (10⁶) expressingthe continuous c-Met IgG4 CAR, the classic CD19 CD8α CAR (adjusted to50% CAR positivity) or mock transduced T cells were infused into NSGmice (n=10 mice per group). Mice were analyzed 60 days followinginfusion by peripheral blood TruCounts to quantify huCD45+ cells per uLof mouse blood. Sample means were not different (two tailed Mann-Whitneyp=0.39); the bars denote S.D.

DETAILED DESCRIPTION

The invention relates to the discovery that particular chimeric antigenreceptors (CARs) transduced into T cells contribute at least toincreased antigen-independent activation of the transduced T cells,increased mean cell volume (MCV) of the transduced T cells, increasedcell population expansion of the transduced T cells, increasedproliferation of the transduced T cells, increased numbers of progeny ofthe transduced T cells, and increased persistence of the transduced Tcell population both in vitro and in vivo. Thus, the invention relatesto compositions and methods for treating cancer, including, but notlimited, to hematologic malignancies and solid tumors, by theadministration of T cells transduced with CARs that contribute toincreased activation and persistence of the transduced T cellpopulation. The present invention relates to a strategy of adoptive celltransfer of T cells transduced to express a chimeric antigen receptor(CAR). CARs are molecules that combine antibody-based specificity for adesired antigen (e.g., tumor antigen) with a T cell receptor-activatingintracellular domain to generate a chimeric protein that exhibits aspecific anti-tumor cellular immune activity.

The invention provides a method for identification of CAR designs thatpermit extensive T-cell proliferation without exogenous cytokineadministration or feeder cells. In one aspect, the invention providescompositions and methods for generating CARs that endow T cells with theability to undergo long-term autonomous proliferation. In one aspect thelong-term proliferation and expansion of CAR T cells are independent ofantigen stimulation and do not require the addition of exogenouscytokines or feeder cells.

In one aspect, the long-term proliferation and expansion of CAR T cellsis partially mediated by constitutive cytokine production. Accordingly,the invention provides a unique molecular signature of CAR T cellshaving a constitutive proliferative phenotype. In one aspect, the uniquemolecule signature of CAR T cells include the expression of one or moreof IFN-γ, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13,Granzyme B and Perforin.

In another embodiment, the invention provides a method of generating aCAR T cell exhibiting a continuous growth phenotype. In one aspect, thecontinuous growth phenotype involves continuous ligand-independentsignal transduction involving canonical TCR and CD28 signal transductionpathways. In another aspect, the continuous proliferation phenotype ofthe CAR T cells can be identified by evaluating the level of scFvsurface expression on the CAR T cells, as CARs expressed brightly at thecell surface sustained proliferation, while CARs expressing at lowerlevel of scFv surface expressing did not exhibit sustained proliferationand cytokine secretion.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Inanother embodiment, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.Preferably, the hinge domain is an IgG4 or CD8α hinge domain.

In various embodiments, the persisting CAR T cells of the invention canbe generated by introducing a lentiviral vector comprising a desired CARthat contributes to at least one of increased antigen-independentactivation of the transduced T cells, increased mean corpuscular volume(MCV) of the transduced T cells, increased cell population expansion ofthe transduced T cells, increased proliferation of the transduced Tcells, increased numbers of progeny of the transduced T cells, andincreased persistence of the transduced T cell population both in vitroand in vivo. By way of example, the CAR of the invention comprises ananti-c-Met, IgG4 hinge, CD28 transmembrane and CD28 and CD3zetasignaling domains. By way of another example, the CAR of the inventioncomprises an anti-mesothelin (SS1), IgG4 hinge, CD28 transmembrane andCD28 and CD3zeta signaling domains. By way of another example, the CARof the invention comprises an anti-mesothelin, CD8a hinge, CD28transmembrane domain and CD28 and CD3zeta signaling domains. By way ofanother example, the CAR of the invention comprises an anti-CD19, IgG4hinge, CD28 transmembrane, and CD28 and CD3zeta signaling domains. Byway of another example, the CAR of the invention comprises an anti-CD19,CD8a hinge domain, CD28 transmembrane and CD28 and CD3zeta signalingdomains. The CAR T cells of the invention are able to replicate in vivoresulting in long-term persistence that can lead to sustained tumorcontrol.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR for the treatment of a patient havingcancer or at risk of having cancer using lymphocyte infusion.Preferably, autologous lymphocyte infusion is used in the treatment.Autologous PBMCs are collected from a patient in need of treatment and Tcells are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of and/or for the testing of the present invention, thepreferred materials and methods are described herein. In describing andclaiming the present invention, the following terminology will be usedaccording to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or in some instances ±10%, or in some instances ±5%,or in some instances ±1%, or in some instances ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. The anantibody in the present invention may exist in a variety of forms wherethe antigen binding portion of the antibody is expressed as part of acontiguous polypeptide chain including, for example, a single domainantibody fragment (sdAb), a single chain antibody (scFv) and a humanizedantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to at least one portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,sdAb (either V_(L) or V_(H)), camelid V_(HH) domains, scFv antibodies,and multi-specific antibodies formed from antibody fragments. The term“scFv” refers to a fusion protein comprising at least one antibodyfragment comprising a variable region of a light chain and at least oneantibody fragment comprising a variable region of a heavy chain, whereinthe light and heavy chain variable regions are contiguously linked via ashort flexible polypeptide linker, and capable of being expressed as asingle chain polypeptide, and wherein the scFv retains the specificityof the intact antibody from which it was derived. Unless specified, asused herein an scFv may have the V_(L) and V_(H) variable regions ineither order, e.g., with respect to the N-terminal and C-terminal endsof the polypeptide, the scFv may comprise V_(L)-linker-V_(H) or maycomprise V_(H)-linker-V_(L).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations, and which normally determines theclass to which the antibody belongs.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations. Kappy (κ) and lambda (λ) light chainsrefer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to encodepolypeptides that elicit the desired immune response. Moreover, askilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid. The term “anti-tumor effect”as used herein, refers to a biological effect which can be manifested bya decrease in tumor volume, a decrease in the number of tumor cells, adecrease in the number of metastases, an increase in life expectancy, oramelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe invention in prevention of the occurrence of tumor in the firstplace.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The terms “microarray” and “array” refers broadly to both “DNAmicroarrays” and “DNA chip(s),” and encompasses all art-recognized solidsupports, and all art-recognized methods for affixing nucleic acidmolecules thereto or for synthesis of nucleic acids thereon. Preferredarrays typically comprise a plurality of different nucleic acid probesthat are coupled to a surface of a substrate in different, knownlocations. These arrays, also described as “microarrays” or colloquially“chips” have been generally described in the art, for example, U.S. Pat.Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 5,800,992, 6,040,193,5,424,186 and Fodor et al., 1991, Science, 251:767-777, each of which isincorporated by reference in its entirety for all purposes. Arrays maygenerally be produced using a variety of techniques, such as mechanicalsynthesis methods or light directed synthesis methods that incorporate acombination of photolithographic methods and solid phase synthesismethods. Techniques for the synthesis of these arrays using mechanicalsynthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261, and6,040,193, which are incorporated herein by reference in their entiretyfor all purposes. Although a planar array surface is preferred, thearray may be fabricated on a surface of virtually any shape or even amultiplicity of surfaces. Arrays may be nucleic acids on beads, gels,polymeric surfaces, fibers such as fiber optics, glass or any otherappropriate substrate. (See U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated byreference in their entirety for all purposes.) Arrays may be packaged insuch a manner as to allow for diagnostic use or can be an all-inclusivedevice; e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated intheir entirety by reference for all purposes. Arrays are commerciallyavailable from, for example, Affymetrix (Santa Clara, Calif.) andApplied Biosystems (Foster City, Calif.), and are directed to a varietyof purposes, including genotyping, diagnostics, mutation analysis,marker expression, and gene expression monitoring for a variety ofeukaryotic and prokaryotic organisms. The number of probes on a solidsupport may be varied by changing the size of the individual features.In one embodiment the feature size is 20 by 25 microns square, in otherembodiments features may be, for example, 8 by 8, 5 by 5 or 3 by 3microns square, resulting in about 2,600,000, 6,600,000 or 18,000,000individual probe features.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention relates to compositions and methods of transducingT cells with chimeric antigen receptors (CARs) that generate apersisting population of T cells that exhibit increasedantigen-independent activation, increased mean cell volume (MCV),increased cell population expansion, increased proliferation, increasednumbers of progeny, induction of constitutive cytokine secretion, andincreased persistence of the transduced T cell population both in vitroand in vivo, as compared with their untransduced counterparts. Thus, thepresent invention includes compositions and methods for treating canceramong other diseases. The cancer may be a hematological malignancy, asolid tumor, a primary or a metastasizing tumor. Other diseasestreatable using the compositions and methods of the invention includeviral, bacterial and parasitic infections as well as autoimmunediseases.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR that contributes to increased activation orproliferation of the transduced T cell and wherein the CAR T cellexhibits an antitumor property. The CAR of the invention can beengineered to comprise an extracellular domain having an antigen bindingdomain fused to an intracellular signaling domain of the T cell antigenreceptor complex zeta chain (e.g., CD3 zeta). The CAR of the inventionwhen expressed in a T cell is able to redirect antigen recognition basedon the antigen binding specificity. Exemplary antigens include cMet,mesothelin and CD19. However, the invention is not limited to targetingcMet, mesothelin and CD19. Rather, the invention includes any antigenbinding moiety that when bound to its cognate antigen, affects a tumorcell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The antigen binding moiety is preferably fusedwith an intracellular domain from one or more of a costimulatorymolecule and a zeta chain. Preferably, the antigen binding moiety isfused with one or more intracellular domains selected from the group ofa CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3-zetasignaling domain, and any combination thereof.

Compositions

The present invention provides chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding moiety. In some embodiment, the extracellulardomain also comprises a hinge domain. The intracellular domain orotherwise the cytoplasmic domain comprises, a costimulatory signalingregion and a zeta chain portion. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

Antigen Binding Moiety

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding moiety. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen of interest by way of engineering a desired antigenbinding moiety that specifically binds to an antigen on a tumor cell. Inthe context of the present invention, “tumor antigen” or“hyperporoliferative disorder antigen” or “antigen associated with ahyperproliferative disorder,” refers to antigens that are common tospecific hyperproliferative disorders such as cancer. The antigensdiscussed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding moiety of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, cMet,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding moiety portion of the CARtargets an antigen that includes but is not limited to cMet, CD 19,CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, cMet, PSMA, Glycolipid F77,EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifCD19 is the desired antigen that is to be targeted, an antibody for CD19can be used as the antigen bind moiety for incorporation into the CAR ofthe invention.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or froman immunoglobulin such as IgG4. Alternatively the transmembrane domainmay be synthetic, in which case it will comprise predominantlyhydrophobic residues such as leucine and valine. Preferably a triplet ofphenylalanine, tryptophan and valine will be found at each end of asynthetic transmembrane domain. Optionally, a short oligo- orpolypeptide linker, preferably between 2 and 10 amino acids in lengthmay form the linkage between the transmembrane domain and thecytoplasmic signaling domain of the CAR. A glycine-serine doubletprovides a particularly suitable linker.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3-zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB. In yetanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

Vectors

The present invention encompasses a DNA construct comprising sequencesof a CAR, wherein the sequence comprises the nucleic acid sequence of anantigen binding moiety operably linked to the nucleic acid sequence ofan intracellular domain. An exemplary intracellular domain that can beused in the CAR of the invention includes but is not limited to theintracellular domain of CD3-zeta, CD28, 4-1BB, and the like. In someinstances, the CAR can comprise any combination of CD3-zeta, CD28,4-1BB, and the like.

In one embodiment, the CAR of the invention comprises an anti-cMet, IgG4hinge domain, and CD28 transmembrane and CD28 and CD3zeta signalingdomains. In another embodiment, the CAR of the invention comprises ananti-mesothelin, IgG4 hinge domain, and CD28 and CD3zeta signalingdomains. In a further embodiment, the CAR of the invention comprises ananti-mesothelin, CD8a hinge domain, and CD28 transmembrane and CD28 andCD3zeta signaling domains. In yet another embodiment, the CAR of theinvention comprises an anti-CD 19, IgG4 hinge domain, and CD28transmembrane and CD3zeta signaling domains. In still anotherembodiment, the CAR of the invention comprises an anti-CD19, CD8a hingedomain, and CD28 transmembrane and CD28 and CD3zeta signaling domains.

In one embodiment, the CAR of the invention comprises the nucleic acidsequence set forth in SEQ ID NO: 1. In another embodiment, the CAR ofthe invention comprises the nucleic acid sequence set forth in SEQ IDNO: 2. In a further embodiment, the CAR of the invention comprises thenucleic acid sequence set forth in SEQ ID NO: 3.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD 14, CD20, CD 11b, CD 16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV). For example, the LV encodes a CAR thatcombines an antigen recognition domain of a specific antibody with anintracellular domain of CD3-zeta, CD28, 4-1BB, or any combinationsthereof. Therefore, in some instances, the transduced T cell can elicita CAR-mediated T-cell response.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises a binding moiety that specifically interacts with apredetermined target, a zeta chain portion comprising for example theintracellular domain of human CD3zeta, and a costimulatory signalingregion.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, it was unexpectedthat the CART 19 cells of the invention can undergo robust in vivo Tcell expansion and persist at high levels for an extended amount of timein blood and bone marrow and form specific memory T cells. Withoutwishing to be bound by any particular theory, CAR T cells maydifferentiate in vivo into a central memory-like state upon encounterand subsequent elimination of target cells expressing the surrogateantigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding moiety in the CAR. For example, a CART19 cells elicits an immuneresponse specific against cells expressing CD19.

While the data disclosed herein specifically disclose lentiviral vectorcomprising anti-CD19 scFv derived from FMC63 murine monoclonal antibody,human CD8α hinge and transmembrane domain, and human 4-1BB and CD3zetasignaling domains, the invention should be construed to include anynumber of variations for each of the components of the construct asdescribed elsewhere herein. That is, the invention includes the use ofany antigen binding moiety in the CAR to generate a CAR-mediated T-cellresponse specific to the antigen binding moiety. For example, theantigen binding moiety in the CAR of the invention can target a tumorantigen for the purposes of treat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARdesigned to target CD19 can be used to treat cancers and disordersincluding but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage postallogeneic bone marrow transplantation, and the like. In anotherembodiment, the CAR can be designed to target CD22 to treat diffuselarge B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogeneic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, the CAR can be designed to target mesothelin to treatmesothelioma, pancreatic cancer, ovarian cancer, and the like. Inanother embodiment, the CAR can be designed to target CD33/IL3Ra totreat acute myelogenous leukemia and the like. In a further embodiment,the CAR can be designed to target cMet to treat triple negative breastcancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR can be designed to target PSMA to treatprostate cancer and the like. In another embodiment, the CAR can bedesigned to target Glycolipid F77 to treat prostate cancer and the like.In a further embodiment, the CAR can be designed to target EGFRvIII totreat gliobastoma and the like.

In one embodiment, the CAR can be designed to target GD-2 to treatneuroblastoma, melanoma, and the like. In another embodiment, the CARcan be designed to target NY-ESO-1 TCR to treat myeloma, sarcoma,melanoma, and the like. In a further embodiment, the CAR can be designedto target MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammalPreferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of CCL. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing CCL. Thus, the present inventionprovides methods for the treatment or prevention of CCL comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “an tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the CARs and methods described herein, or other methodsknown in the art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Constitutive CAR T Cell Proliferation

Adoptive immunotherapy has potent antitumor effects that are dependenton engraftment and proliferation of the transferred T cells in the host.The results presented herein demonstrate that certain chimeric antigenreceptors (CARs) endow T cells with the ability to undergo long-termautonomous proliferation. Transduction of human T cells with secondgeneration CARs encoding the CD28 and CD3ζ endodomains resulted insustained proliferation for up to three months following a singlestimulation through the TCR. This numeric expansion was independent ofantigen stimulation and did not require the addition of exogenouscytokines or feeder cells. Both gene array and functional assays haveidentified that the prolonged growth is partially mediated byconstitutive cytokine production. Microarray analysis identified aunique molecular signature of CAR T cells with the constitutiveproliferative phenotype. Sustained expression of the endogenous IL-2locus has not previously been reported in primary T cells. The CD28 andCD3ζ endodomains appear critical as constitutive signaling through NFkB,Akt, Erk and NFAT is observed. Further, not all CARs that signal throughCD28 and CD3ζ could sustain ligand independent T-cell proliferation. Thepropagated CAR+ T cells retain a diverse TCR repertoire andtransformation was not observed. The density of CAR expression at thecell surface is an important variable in determining whether the CAR hasa constitutive or inducible growth phenotype. The identification of CARdesigns that permit extensive T-cell proliferation without exogenouscytokine administration or feeder cells may have implications to eitherexploit or avoid CARs with constitutive activity.

The materials and methods used in this example are now described.

Materials and Methods

Construction of Lentiviral Vectors with Differing Eukaryotic Promotersand CARs

FIG. 1A shows schematic diagrams of the CARs used in this study. AllCARs contain a scFv that recognizes either the human CD19, mesothelin orc-Met antigen. Lentiviral vectors from previously published work wereused to encode the anti-CD19 FMC63 CD8α (Tammana et al., 2010, Hum GeneTher 21:75-86), the anti-mesothelin SS1 CD8α, and the anti-mesothelinSS1 CD8α Δtail CAR constructs (Carpenito et al., 2009, Proc Natl AcadSci USA 106:3360-3365). The c-Met 5D5 IgG4 construct was used as atemplate to generate the SS1 IgG4 and CD19 IgG4 CAR constructs throughPCR splicing and overlap extension. Restriction sites were introducedvia PCR primers, which allowed for cloning into third generationself-inactivating lentiviral plasmids. The cytomegalovirus (CMV) andelongation factor-1α (EF-1α) promoter sequences were amplified via PCRfrom previously constructed plasmids and introduced into pre-existingCAR containing constructs (Milone et al., 2009, Mol Ther 17:1453-1464)using standard molecular biology techniques. Representative CARs aredepicted below.

cMet IgG4 28z (SEQ ID NO: 1)atgctgctgctggtgaccagcctgctgctgtgtgagagccccaccccgcctttctgctgatccccgacatccagatgacccagagccccagcagcgtgagcgccagcgtgggcgaccgggtgaccatcacctgccgggccagccagggcatcaacacctggctggcctggtatcagcagaagcccggcaaggcccccaagagagatctacgccgccagcagcctgaagagcggcgtgcccagccggtttagcggctctggctctggcgccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcaggccaacagcttccccctgacctttggcggcggaacaaaggtggagatcaagggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggccaggtgcagctggtgcagagcggagccgaggtgaagaagcctggcgcctccgtcaaggtgtcctgcgaggccagcggctacaccttcaccagctacggcttcagagggtgcggcaggcaccaggccagggcctcgaatggatgggctggatcagcgccagcaacggcaacacctactacgcccagaagagcagggcagggtcaccatgaccaccgacaccagcaccagcagcgcctacatggaactgcggagcctgagaagcgacgacaccgccgtgtactactgcgccagggtgtacgccgactacgccgattactggggccagggcaccctggtgaccgtgagcagcgagagcaagtacggccctccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagccggacccccgaggtgacctgtgtggtggtggacgtgtcccaggaggaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagttcaatagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgtaaggtgtccaacaagggcctgcccagcagcatcgagaaaaccatcagcaaggccaagggccagcctcgggagccccaggtgtacaccctgccccctagccaagaggagatgaccaagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagccggtggcaggagggcaacgtctttagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtccctgggcaagatgttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtgaggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggatttcgccgcctaccggagccgggtgaagttcagccggagcgccgacgcccctgcctaccagcagggccagaaccagctgtacaacgagctgaacctgggccggagggaggagtacgacgtgctggacaagcggagaggccgggaccctgagatgggcggcaagccccggagaaagaacccccaggagggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtaccagggcctgagcaccgccaccaaggatacctacgacgccctgcacatgcaggccctgccccccagatgaSS1 IgG4 28z (SEQ ID NO: 2)atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaaagcagcgagagcaagtacggccctccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagccggacccccgaggtgacctgtgtggtggtggacgtgtcccaggaggaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagttcaatagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgtaaggtgtccaacaagggcctgcccagcagcatcgagaaaaccatcagcaaggccaagggccagcctcgggagccccaggtgtacaccctgccccctagccaagaggagatgaccaagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagccggtggcaggagggcaacgtctttagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtccctgggcaagatgttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtgaggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggatttcgccgcctaccggagccgggtgaagttcagccggagcgccgacgcccctgcctaccagcagggccagaaccagctgtacaacgagctgaacctgggccggagggaggagtacgacgtgctggacaagcggagaggccgggaccctgagatgggcggcaagccccggagaaagaacccccaggagggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtaccagggcctgagcaccgccaccaaggatacctacgacgccctgcacatgcaggccctgccccccagatga CD19 IgG4 28z (SEQ ID NO: 3)atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaagcagcgagagcaagtacggccctccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagccggacccccgaggtgacctgtgtggtggtggacgtgtcccaggaggaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagttcaatagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgtaaggtgtccaacaagggcctgcccagcagcatcgagaaaaccatcagcaaggccaagggccagcctcgggagccccaggtgtacaccctgccccctagccaagaggagatgaccaagaaccaggtgtccctgacctgcctggtgaagggatctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggcagatcttcctgtacagccggctgaccgtggacaagagccggtggcaggagggcaacgtattagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtccctgggcaagatgttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtgaggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggatttcgccgcctaccggagccgggtgaagttcagccggagcgccgacgcccctgcctaccagcagggccagaaccagctgtacaacgagctgaacctgggccggagggaggagtacgacgtgctggacaagcggagaggccgggaccctgagatgggcggcaagccccggagaaagaacccccaggagggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtaccagggcctgagcaccgccaccaaggatacctacgacgccctgcacatgcaggccctgccccccagatga

Microarray Studies

Sample Collection. Human CD4+ T cells from three normal donors werestimulated and transduced with either the c-Met IgG4 or CD19 CD8α CARconstruct. Cell pellets were collected and frozen on day 0 prior tostimulation, day 6 and day 11 at rest down for all samples and 24 forthe c-Met IgG4 CAR.

Microarray Target Preparation and Hybridization. Microarray serviceswere provided by the UPenn Microarray Facility, including qualitycontrol tests of the total RNA samples by Agilent Bioanalyzer andNanodrop spectrophotometry. All protocols were conducted as described inthe Affymetrix GeneChip Expression Analysis Technical Manual. Briefly,100 ng of total RNA was converted to first-strand cDNA using reversetranscriptase primed by poly(T) and random oligomers that incorporatedthe T7 promoter sequence. Second-strand cDNA synthesis was followed byin vitro transcription with T7 RNA polymerase for linear amplificationof each transcript, and the resulting cRNA was converted to cDNA,fragmented, assessed by Bioanalyzer, and biotinylated by terminaltransferase end labeling. cRNA yields ranged from 36-89 μg, and cDNA wasadded to Affymetrix hybridization cocktails, heated at 99° C. for 5 minand hybridized for 16 h at 45° C. to Human Gene 1.0ST GeneChips(Affymetrix Inc., Santa Clara Calif.). The microarrays were then washedat low (6×SSPE) and high (100 mM MES, 0.1M NaCl) stringency and stainedwith streptavidin-phycoerythrin. A confocal scanner was used to collectfluorescence signal after excitation at 570 nm.

Initial Data Analysis. Affymetrix Command Console and Expression Consolewere used to quantify expression levels for targeted genes; defaultvalues provided by Affymetrix were applied to all analysis parameters.Border pixels were removed, and the average intensity of pixels withinthe 75th percentile was computed for each probe. The average of thelowest 2% of probe intensities occurring in each of 16 microarraysectors was set as background and subtracted from all features in thatsector. Probe sets for positive and negative controls were examined inExpression Console, and Facility quality control parameters wereconfirmed to fall within normal ranges. Probes for each targeted genewere averaged and inter-array normalization performed using the RMAalgorithm.

Analysis of Terminal Telomeric Restriction Fragment Lengths

Telomeric restriction fragment length analysis was performed essentiallyas described (Lukens et al., 2009, Alzheimers Dement 5:463-469).Briefly, 2 μg of genomic DNA was digested with RsaI+HinfI and resolvedon a 0.5% agarose gel, which was then dried and probed with a³²P-labeled (CCCTAA) 4 oligonucleotide. After washing, the samples werevisualized with a Phosphor imager.

Statistical Analysis

Raw data obtained from microarray core was normalized with robustmultichip average (RMA). Analysis was performed using a 3-way mixedmodel ANOVA with factors being sample date, treatment group and donorID. An interaction term between sample and collection date was added. Inconjunction with the multiple pair wise contrasts that were looked at ap-value and fold change were determined. For all p-values we calculatedthe FDR corrected p-value using the method of Benjamini and Hochberg asimplemented by Partek Genomic Suite (Partek). For transcription factorand cytokine dot plots the normalized absolute log(2) gene expressionintensities were plotted. Cluster analysis was performed using Euclideandistance of median normalized log(2) gene expression intensities withaverage linkage. All growth curves, MFI and engraftment plots wereplotted using Prism (GraphPad Software). All error bars arerepresentative of standard deviation. A two tailed Mann-Whitney test wasperformed for the in vivo engraftment studies.

Cell Lines and Culture

Blood samples were obtained from the Human Immunology Core of theUniversity of Pennsylvania where peripheral blood CD4+ T cells werenegatively isolated using RosetteSep Kits (Stem cell Technologies).Cells were cultured in R10 (RPMI 1640 media supplemented with 10% FCS,100-U/ml penicillin, 100 μg/m streptomycin sulfate, 10 mM Hepes) in a37° C. and 5% CO₂ incubator. For stimulation, CD4+ T cells were culturedwith activating beads coated with antibodies to CD3 and CD28 at a 1:3cell to bead ratio. Cells were transduced with lentiviral vectorscontaining CAR constructs approximately 24 hrs following stimulation. Tcells were monitored, kept at a concentration of 0.75×10⁶/mL and wereconsidered rested when MCV <175. The M30 and NCI-H522 tumor lines wereused in cell killing assays. The M30 cell line (Crisanti et al., 2009,Mol Cancer Ther 8:2221-2231) is a mesothelial tumor derived at theUniversity of Pennsylvania from mesothelioma tumor tissues fromindividual patients and was cultured in E-media (10% FCS, 1× ITES, 10 mMHEPES, 0.5 mM Na Pyruvate, 0.1 mMMEM NEAAs, 100 ug/mL Pen/Strep, 1 ng/mLEGF, 18 ng/mL HC, 0.1 nM T3 in RPMI) while the NCI-H522 (adenocarcinoma)was obtained from the National Cancer Institute and cultured in R10.Jurkat cell line stably transfected with a plasmid containing d2EGFPunder the control of a minimal promoter bearing the NFAT consensusbinding sequence (pNFAT-d2EGFP) was kindly provided by Arthur Weiss(University of California at San Francisco).

Flow Cytometry and Antibodies

CAR surface staining was performed in FACS buffer (PBS with 3% fetalcalf serum) using biotin conjugated polyclonal antibody (JacksonImmunoResearch). Rabbit anti-human IgG (H+L) was used for cMet IgG4, SS1IgG4, and CD19 IgG4, while goat anti-mouse (Fab′)2 was used as primaryfor SS1 CD8a, CD19 CD8a, and SS1 Δtail. Secondary stain for CAR was doneusing streptavadin-APCeFluor780 (eBioscience). Cell surface markeranalysis was done using CD25 PerCp-Cy5.5 (eBioscience, clone BC96), CD70PE (BD, clone Ki-24), PD-1 PerCP-eFluor710 (eBioscience, clone J105),CD45RO eFluor450 (eBioscience, clone UCHL1), CD27 v450 (BD, cloneM-T271), CD28 FITC (eBioscience, clone CD28.2), CD62L PE (eBioscience,clone DREG-56), CCR7 FITC (BD, clone 150503), Crtam APC (Biolegend,clone Cr24.1) and c-Met PE (R&D systems, clone 95106) at the recommendedconcentrations. c-Met antigen staining was done using monoclonalanti-human HGF We-MET-PE (R&D, clone#95106), and mesothelin expressionwas analyzed with primary monoclonal mouse anti-human CAK1 (Covance) at1:50 followed by polyclonal goat anti-mouse PE (BD) at 1:100. Sampleswere analyzed on a LSR II (BD) and analyzed with FlowJo software(TreeStar).

PhosFlow was performed on days 6, 10 and 25. Cells were fixed using BDcytofix buffer (BD) for 10 min at 37 C followed by permeabilizationusing BD Phosflow Perm Buffer III (BD) at 4 C for 30 minutes. Cells werestained at RT for 30 min in the dark using PE anti-Erk1/2 (pT202/pY204)(BD, clone 20A), PE conjugated anti-Akt (pS473) (BD, clone M89-61), PEconjugated anti-NF-kB p65 (pS529) (BD, clone K10-895.12.50), or PEconjugated anti-S6 (pS235/pS236) (BD, clone N7-548) at manufacturesrecommended concentrations. Positive controls were samples from eachgroup stimulated for 10 min using PMA/Ionomycin prior to fixation, whilenegative controls were fully stimulated T cells stained using PEconjugated IgG2b kappa isotype control (BD, clone 27-35). Samples wererun on a LSR II (BD Bio-sciences) and analyzed with FlowJo software(TreeStar).

Cytokine Measurements

CD4+ T cells were transduced with CAR constructs as described elsewhereherein. On days 6, 10, and 30 one million cells were taken from eachgroup, pelleted, washed in R10 and plated at 1×10^6/mL in fresh media.At 24 hrs supernatant was collected and frozen at −80° C. Quantificationof soluble cytokine factors was performed using Luminex bead arraytechnology and kits purchased from Life Technologies (Invitrogen30-plex). Assays were performed as per the manufacturer protocol with9-point standard curve generated using a 3-fold dilution series andaccording to laboratory SOP. Each sample was evaluated in duplicate at1:3 dilution; calculated % CV for the duplicate measures was in mostcases less than 5% and always less than 15%. Data were acquired on aFlexMAP-3D and analyzed using XPonent 4.0 software and 5-parameterlogistic regression analysis. Standard curve quantification ranges weredetermined by the 80-120% (observed/expected value) range.

Conditioned Media Transfer

Supernatant from c-Met IgG-4 transduced T cell cultures was collected onday 56, filtered through a 70 μm filter and frozen at −80° C. in 10 mLaliquots. Day 56 media was thawed and added to unstimulated naïve CD4+ Tcells in culture to reach a final concentration of 12.5%, 25%, and 50%c-Met IgG4 supernatant relative to starting media. As controls, mediawith and without 100 IU of IL-2 was also included, as well as CD3/CD28bead stimulated cells kept in culture with initial stimulation on day 0and re-stimulation on day 12. Mean cell volumes were determined and cellmedia was readjusted every two days to maintain IL-2 concentrationwithin control group and appropriate c-Met IgG4 media transfer ratiodescribed elsewhere herein.

Vβ Diversity Determination

CD4+ human T cells were isolated, stimulated and transduced with c-MetIgG4 CAR as described elsewhere herein. Donor matched untransduced cellswere stimulated and expanded simultaneously as control. Untransducedcontrols required two additional bead stimulations to maintain inculture. Cells were cryopreserved at D0, D13 and D34. Cells were thawedsimultaneously and allowed to rest overnight. TCR Vβ analysis wasperformed using the IOTest Beta Mark TCR V kit (Beckman Coulter) whichcontains directly-conjugated antibodies specific for the following Vβfamilies: 1, 2, 3, 4, 5.1, 5.2, 5.3, 7.1, 7.2, 8, 9, 11, 12, 13.1, 13.2,13.6, 14, 16, 17, 18, 20, 21.3, 22, and 23. Samples were run on a LSR II(BD) with subsequent analysis in FlowJo (TreeStar) to determine percentof total population.

Cytotoxicity Assay

A mix of CD4+ and CD8+ human T cells electroporated with mRNA encodingthe indicated CAR were used for in vitro killing. CD19 CD8α and c-MetIgG4 CARs were subcloned into a pGEM.64A-based vector previouslydescribed (Zhao et al., 2006, Mol Ther 13:151-159). The SS1 CD8α CARmRNA was made as described (Zhao et al., 2010, Cancer Res 70:9062-9072).The replaced CAR cDNAs were confirmed by direct sequencing andlinearized by SpeI digestion prior to RNA IVT. mScript RNA System(Epicentre, Madison, Wis.) was used to generate capped IVT RNA. The IVTRNA was purified using an RNeasy Mini Kit (Qiagen, Inc., Valencia,Calif.), and purified RNA was eluted in RNase-free water at 1-2 mg/ml.Human T cells were stimulated by CD3/CD28 beads as described (Carpenitoet al., 2009, Proc Natl Acad Sci USA106:3360-3365). On day 0 thestimulated T cells were washed three times with Opti-MEM and resuspendedin Opti-MEM at the final concentration of 1-3×10⁸/ml prior toelectroporation. Subsequently, the stimulated T cells were mixed with 10μg/0.1 ml of IVT RNA (as indicated) and electroporated in a 2-mm cuvette(Harvard Apparatus BTX, Holliston, Mass.) using an ECM830 Electro SquareWave Porator (Harvard Apparatus BTX). Tumor lines were then harvestedwith trypsin and plated in a 6 well dish at 0.2×10^6/mL. 24 hours post Tcell electroporation and tumor plating T cells were combined with targetcells at increasing effector:target (E:T) ratios in a 6 well plate aswell, alongside a no T cell control. Cells were incubated at 37° C. for18 hrs. Cells were collected after incubation, wells were re-trypsinizedand washed repeatedly to collect all tumor and T cells. Cells mixtureswere stained for tumor with anti-EpCAM (BD, clone EBA-1), T cells withanti-CD45 (BD, clone 2D1), and with 7-AAD (Invitrogen). Cells wereresuspended in 400 uL FACS buffer containing counting beads (Invitrogen)to normalize data acquisition across samples. Samples were then filteredthrough a 35 μm filter (BD Falcon) and put on ice for analysis. Cellswere run on a LSR II (BD) and collection was performed by collecting1500 bead events for all samples. Analysis was performed by gating onEpCAM(+), CD45(−), and 7-AAD(−) cells in FlowJo (TreeStar). Percentlysis was calculated by dividing total live cells in no T cell controlgroup, by each experimental condition of increasing E:T ratio.

In Vivo T Cell Persistence Experiments

All animal experiments were approved by the University of PennsylvaniaInstitutional Animal Care and Use Committee. NSG mice(NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ) were used for engraftment andpersistence experiments. The mice were housed under specificpathogen-free conditions in microisolator cages and given unrestrictedaccess to autoclaved food and acidified water. Animals of both sexeswere used for experiments at approximately 20 weeks of age. Human CD4+ Tcells were isolated, stimulated and transduced as previously described.A total of 10×10⁶ cells/mouse were injected peripherally by tail veininjections of which 50% were CAR (+) in the c-Met IgG4 group. Peripheralbleeds were done after 60 days and TruCounts (BD) were done usinganti-human CD45 APC-H7 staining for absolute quantification. Sampleswere analyzed on a LSR II (BD Bioscience) and quantification wasperformed using FlowJo (TreeStar).

DNA Isolation and Q-PCR Analysis

Q-RT/PCR analysis: RNA was isolated from cell lines using RNAqueous RNAisolation kits (Ambion), and cDNA synthesized using iScript cDNAsynthesis kits (Bio-Rad). Samples were analyzed for expression of c-met,mesothelin, and PP1B (housekeeping transcript) using ABI Taqman-basedtechnologies and the following ABI recommended gene specific primerprobe sets which span exon/intron boundaries: c-met: Hs01565584_m1*;mesothelin: HS00245879_m1*, and PP1B: Hs00168719_m1*. All amplificationreactions were performed using an ABI 7500 FAST instrument (ABI-Lifetechnologies), and established laboratory protocols. Each transcript wasevaluated in triplicate. Ct values for each amplification reaction weredetermined using pre-established assay-specific threshold values, with aminimum of 2/3 replicates with % CV <15% required to record a Ct value.Average Ct values were calculated and reported. RQ (relativequantification) values for each transcript was determined according tothe formula: RQ=2−−ΔCt, with −ΔCt=−ΔCtsample−−ΔCtreference, with−ΔCtsample=Ctsample−Ctsample normalizer and−ΔCtreference=Ctreference−Ctreference normalizer (Pfaffl, 2001, NucleicAcids Res 29(9):e45). For all analyses, the ovarian carcinoma cell lineOV79 (positive for both MAGE-A3) served as the reference sample. Theovarian carcinoma-derived cell line OV-79 has been previously described(Bertozzi et al., 2006, In Vitro Cell Dev Biol Anim 42(3-4):58-62).

The results of this experimental example are now described.

Construction and Characterization of Chimeric Antigen Receptors

A plethora of CARs have been generated that express CD28 and CD3ζdownstream of antibody fragments that mediate surrogate antigenrecognition (Geiger et al., 2001, Blood 98:2364-2371; Arakawa et al.,2002, Anticancer Research 4285-4289; Haynes et al., 2002, J Immunol169(10):5780-6; Maher et al., 2002, Nature Biotechnology 20:70-75;Finney et al, 2004, J Immunol 172:104-113; Gyobu et al., 2004, CancerRes 64:1490-1495; Moeller et al., 2004, Cancer Gene Ther 11:371-379;Teng et al., 2004, Hum Gene Ther 15:699-708; Friedmann-Morvinski et al.,2005, Blood 105:3087-3093; Pule et al., 2005, Molecular Therapy12:933-941; Westwood et al., 2005, Proc Natl Acad Sci USA102:19051-19056; Willemsen et al., 2005, J Immunol 174:7853-7858;Kowolik et al, 2006, Cancer Res 66:10995-11004; Loskog et al., 2006,Leukemia 20:1819-1828; Shibaguchi et al., 2006, Anticancer Res26:4067-4072; Teng et al., 2006, Human Gene Therapy 17:1134-1143;Brentjens et al., 2007, Clin Cancer Res 13:5426-5435). Given that thesetransgenes were constructed differently and by different investigatorsat different institutes, it remains unknown how these CARs would performwith a common expression system and a standardized culture system thathas been optimized for clinical use. Therefore, a set of CARs targetingc-Met, mesothelin and CD19 was expressed in primary human CD4+ T cells(FIG. 1A). The CARs encoded IgG4 or CD8α hinge domains, CD28 or CD8αtransmembrane domains and the signaling domains were comprised of CD28and CD3ζ. A CAR with a truncated signaling domain, and a CD19 4-1BB:CD3ζCAR used in a previous clinical trial (Porter et al., 2011, N Engl J Med365:725-733) served as controls. All CARs were expressed constitutivelyusing an EF-1α promoter, and in a typical experiment 50% of the cellsinitially expressed the CAR and had similar levels of expression on thesurface by day 6 after transduction (FIG. 1B). The c-Met CAR T cells hadspecific and potent cytotoxicity (FIG. 8), and previous studies haveshown that the CARs specific for CD 19 and mesothelin have similarlypotent effector functions (Carpenito et al., 2009, Proc Natl Acad SciUSA 106:3360-3365; Milone et al., 2009, Mol Ther 17:1453-1464).

Chimeric Antigen Receptors with CD28 and CD3ζ can Induce Constitutive TCell Proliferation

Previous studies suggested that antitumor effects after CAR T cellinfusions require sustained expansion of CAR T cells in vivo afteradoptive transfer (Kalos et al., 2011, Sci Transl Med 3:95ra73). Todetermine the proliferative capacity of the CART cells, CD4+ T cellswere activated with anti-CD3 and CD28 beads, transduced with thelentiviral vector encoding the CAR and then propagated without furtherstimulation in the absence of exogenous cytokines or feeder cells.Unexpectedly, constitutive proliferation of some of the CART cellpopulations was observed (FIG. 2A, left). Exponential growth wasobserved for 60 to 90 days in CAR T cells transduced with the c-Met IgG4construct that encoded CD28 and CD3ζ signaling domains (FIGS. 2A and2B). Similarly, the T cells expressing the anti-mesothelin SS1:IgG4 andSS1:CD8α CARs that signaled through chimeric CD28 and CD3ζ domains alsohad sustained proliferation that was independent of supplementation withexogenous growth factors. Long-term proliferation of CD8+ T cells thatwas independent of antigen stimulation and did not require the additionof exogenous cytokines or feeder cells was also observed (FIG. 2C).However, in order to minimize experimental variables, the rest of theexperiments in this study were carried using bulk CD4+ T cells.

In contrast, the cultures with the other CAR T cell populations had aninitial period of exponential proliferation at the same rate, and afterday 10, a decreasing rate of growth followed by death of the culturewithin 20 days (FIGS. 2A and 2B). For simplicity and clarity the CARconstructs that induce constitutive proliferation are henceforthreferred to as “continuous CARs”, while the CARs that exhibit inducibleproliferation similar to previous reports are referred to as “classicCARs”.

The mean cell volumes were monitored at frequent intervals as a measureof metabolic status and cell cycle (FIG. 2A, right). All T cell culturestransduced with the various CAR constructs increased from a resting (G0)cell volume of ˜190 fl to nearly 600 fl by day 6 of culture, consistentwith the induction of DNA synthesis and the exponential increase in cellnumbers. However, the classic CAR T cells and non-transduced T cellsrapidly returned to a resting cell volume, while the continuous CAR Tcells (c-Met IgG4, SS1 IgG4 and SS1 CD8α) failed to return to a restingcell volume, consistent with the continued cellular proliferation. Onday 20 of culture, the mean cell volume in the cultures of continuousCARs and classic CARs was ˜400 fl and 180 fl, respectively. Notably, thelong term proliferation of the CAR T cells was ligand independent,because the surrogate ligands cMet and mesothelin are not expressed atdetectable levels on the surface of activated human CD4+ T cells (FIG.9), consistent with previous reports (Skibinski et al., 2001, Immunology102:506-514). Q-PCR analysis did not detect transcripts for mesothelinor c-Met in resting CD4 T cells. However, activated T cells, either mocktransduced or transduced with a continuous CAR and cultured under theconditions that lead to long term growth expressed very low butdetectable transcripts specific for c-Met, while mesothelin transcriptsremained undetectable. Given that both c-Met and mesothlelin-specificCARs displayed the continuous growth phenotype, the low level of c-Metexpression in activated T cells is unlikely to be necessary for thesustained growth of the T cells. In addition, the absence of fratricidein the cultures is consistent with ligand-independent continuous growth.

Both continuous and classic CARs migrated at the predicted size asdetermined by Western blots probed with an anti-CD3ζ antibody. The CARsencoding the longer IgG4 hinge migrated more slowly than the CARsencoding CD8α hinges (FIG. 10). Under non-reducing conditions, theseCARs exist as homodimers and monomers. The continuouscytokine-independent polyclonal CD4+ T cell proliferation mediated bythe CD28:CD3ζ CARs was independent of the specificity of the endogenousTCR, and was not the result of clonal outgrowth because the T cellpopulations remained diverse during culture (FIG. 11). Finally the aboveresults were reproducible on T cells obtained from at least 10 differenthealthy donors.

Constitutive Expression of IL-2 and a Diverse Array of Cytokines andChemokines

Without wishing to be bound by any particular theory, it is believedthat the observation of CARs being able to mediate long termconstitutive proliferation of primary T cells has not previously beenreported. To begin to understand the mechanism of this phenomenon,experiments were designed to determine the levels of various cytokinesand other immune-related factors in the supernatants from the culturesthat might be sustaining their unusual longevity in culture. Analysis atthe protein level revealed that the culture supernatants from continuousCARs contained high levels of cytokines characteristic of both Th1 andTh2 CD4+ T cells (FIG. 3A). In contrast, the cultures of classic CAR Tcells had low levels of cytokines that decreased with time of culture.The differences were large in magnitude, as the cytokine concentrationsin the cultures of continuous CARs were 100 to >1000-fold higher thanthe concentrations in the classic CAR cultures. The cytokines likelycontributed to the proliferation because transfer of day 56 conditionedmedium from continuous CAR T cell cultures induced activation ofunstimulated naïve CD4+ T cells (FIG. 3B). These results were confirmedat the transcriptional level, with prominent expression of transcriptsfor IFN-γ, TNF-α, IL-2, IL-4, IL-13, IL-3 and GM-CSF in the cellsisolated from the constitutively proliferating CAR T cells compared tothe classic CAR T cells (FIG. 4A). Consistent with this finding, it wasobserved that continuous CAR T cells outgrew normal T cells in culturesthat were initially comprised of mixtures of CAR T cells and T cellsthat did not express the CAR (FIG. 5A). In addition to the sustainedtranscription and secretion of cytokines and chemokines, the continuousCAR CD4+ T cells had elevated levels of granzyme B and perforin (FIG.4A), consistent with the potent cytotoxic effector function that wasobserved (FIG. 8) and previously reported (Carpenito et al., 2009, ProcNatl Acad Sci USA 106:3360-3365).

Molecular Signature of Constitutive CAR T Cell Proliferation

To further investigate the mechanism of the long term CAR T cellproliferation, experiments were designed to performed gene arrayanalysis. The molecular signature of key transcription factors and genesinvolved in T cell polarization, growth and survival is shown in FIG.4B. The master transcription factors T-bet (TBX21), Eomes, and GATA-3were induced and maintained at high levels in the continuous CAR CD4+ Tcells. In contrast, FoxP3 and RORC were expressed in continuous CAR Tcells at comparable levels to untransduced activated T cells and T cellswith the transient T cell proliferative phenotype. As early as day 11,Bcl-xL was highly expressed in the continuous CART cells compared to theclassic CAR and other control T cell populations (p<0.001), suggestingthat resistance to apoptosis as well as enhanced proliferationcontributes to the long term proliferation of CAR T cells. ContinuousCAR T cells also maintained low level expression of KLRG1, a gene oftenexpressed in terminally differentiated and senescent CD4+ T cells(Voehringer et al., 2002, Blood 100:3698-3702), further emphasizingtheir proliferative capacity.

Hierarchical clustering analysis of the microarray data set indicatesthat the CAR T cells with constitutive T cell proliferation have aunique molecular signature (FIG. 6). It is notable that by day 11, cMetIgG4 CART cells with the long term growth phenotype closely cluster inthe dendrogram. In contrast, naïve T cells were most closely related tountransduced T cells and classic CARs with un-sustained growthphenotypes on day 11 of culture (FIG. 6A). Similarly, fully activatedday 6 T cells from all groups cluster together, while T cells expressingthe continuous CAR constructs diverge by day 11 to display a unique RNAsignature that differs from the genes expressed in untransduced orclassic CART cells on day 6 (FIG. 6B).

The differentially expressed genes in the continuous CAR (c-Met IgG4)and classic CAR (CD19 CD8α) T cells were plotted as a heat map to depictthe relationship of the two populations (FIG. 6C). When analyzed using astringent 5-fold cutoff on day 11 of culture, 183 genes were upregulatedand 36 genes were down regulated in continuous CARs compared to theclassic CAR T cells. Most notably the continuous CAR T cells areenriched for genes related to control of the cell cycle and a diversegroup of cytokines.

Constitutive Induction of Signal Transduction by Continuous CARs

To further investigate the mechanisms of the continuous CAR dependentand ligand-independent T cell growth, experiments were designed tointerrogate the canonical signal transduction pathways that areimplicated in T cell activation and growth (FIG. 5B). T cells expressingclassic or continuous CARs had similar levels of phosphorylation on Aid,ERK1/2, NF-κB p65 (RelA) and S6 on day 6 of culture. In contrast, onlythe continuous CAR T cells had sustained activation of Akt pS473, ERK1/2pT202 and pY204, and RelA pS529 at days 10 and 25 of culture. However,the expression of continuous CARs in cells had only a minor effect on S6pS240 phosphorylation, indicating that the CARs do not lead to universalactivation of T cell signaling pathways. The constitutive signaltransduction together with the above results demonstrating sustainedcytokine secretion suggest that both cell intrinsic and extrinsiceffects of the CAR can lead to the long term expansion of primary humanT cells.

In the above experiments, primary human T cells were subjected to asingle round of activation with anti-CD3 and CD28 beads, and thenfollowed in culture without the addition of exogenous cytokines. Thismethod of culture was chosen because it has been used in clinicaltrials, and the initial activation is necessary to mediate highefficiency CAR expression. To determine if the initial activation of theT cells by anti-CD3 and anti-CD28 signaling is required for thesubsequent constitutive signaling by the CARs, we expressed the variousCARs in a Jurkat T cell line that stably expresses GFP under the controlof the NFAT promoter (FIG. 12). The cells were analyzed 3 days aftertransduction, and only the continuous CARs as classified by the growthphenotype in primary T cells, led to constitutive NFAT activation inJurkat cells. This effect was cell intrinsic as only the Jurkat cellsthat expressed CARs on the surface had GFP expression. In contrast,expression of classic CARs (SS1 CAR with a truncated cytosolic domainand the CD19 CARs) did not lead to constitutive NFAT activation inJurkat cells.

Level of Surface Expression Contributes to Classic or Continuous CAR TCell Phenotype

It has been shown that CARs expressed under the control of differenteukaryotic promoters in primary T cells had widely varying levels ofsurface expression (Milone et al., 2009, Mol Ther 17:1453-1464). Todetermine if the level of surface expression contributed to thecontinuous CAR phenotype, CARs were expressed using the EF-1α or CMVpromoter, resulting in a higher or lower expression (FIG. 7A). The c-MetCAR displayed a continuous phenotype when under the control of EF-1α(FIGS. 7B and 7C). In contrast, the same CAR reverted to a classic CARphenotype when expressed under the control of the CMV promoter,resulting in approximately a 5-fold reduction in surface expression.However, without wishing to be bound by any particular theory, it isbelieved that bright surface expression may not be sufficient for thecontinuous CAR phenotype. Thus high levels of expression at the cellsurface may be necessary but are not sufficient for a continuous CARphenotype.

Continuous CARs Induce T Cell Differentiation and Proliferation withoutTransformation

Polychromatic flow cytometry was used to further characterize the CAR Tcells with constitutive proliferation. The expression of T cellmolecules associated with activation and differentiation was examined oncultures of cells expressing or not expressing the CAR (FIG. 13).Additionally, untransduced T cells were followed over time after asingle round of stimulation with anti-CD3 and CD28 beads (FIG. 14). Theresults show that a progressive enrichment for CAR T cells was observed,so that by day 23 of culture, essentially all cells expressed the CAR.This was associated with bright expression of CD25 at all times on theCAR T cells, whereas CD25 became undetectable by day 14 in thenon-transduced companion control culture (FIG. 14). Similarly, CD70 wasexpressed at progressively higher frequencies in the CART cell culture,a feature not observed in the control culture. In contrast, CD27, theligand for CD70, was expressed in the control cultures, while CD27progressively decreased in the CAR T cell cultures. CD28, CD62L and CCR7expression was maintained in the control cultures while many of thecontinuous CAR T cells became dim or negative for these molecules. Incontrast, PD-1 was transiently expressed in the control cultures at day6, while the CAR T cells had a prominent subpopulation of cells thatretained expression of PD-1. Finally, Crtam, a molecule associated withcell polarity regulation (Yeh et al., 2008, Cell 132:846-859), wasinduced in the continuous CAR T cell cultures and expression of Crtamwas notably restricted to the T cells expressing CARs at the surface.

The potential for the CAR T cells to transform was assessed byobservation of long term cultures in vitro and by transfer of CAR Tcells to immunodeficient mice. The long term cultured CAR T cells do nothave constitutive expression of telomerase, as assessed by hTERTexpression (FIG. 4B), and telomere length decreases with time incultures of continuous CART cells (FIG. 15). In contrast, transformedhuman T cells have been reported to have constitutive telomeraseactivity (Hsu et al., 2007, Blood 109:5168-5177). To date, in more than20 experiments, transformation has not been observed with T cellstransduced with continuous CARs.

As a potentially more sensitive assay to detect the potential fortransformation, NSG (NOD-SCID-γc−/−) mice were used, as previous studieshave shown that adoptively transferred transformed and malignant T cellscan form tumors in immunodeficient mice (Newrzela et al., 2011, Mol Med17:1223-1232). Groups of mice were infused with fully activated T cellsor with continuous CAR T cells and proliferation assessed byquantification of T cells in the mice and effector function assessed bythe induction of xenogeneic graft versus host disease in the mice (FIG.16). By day 60, xeno-reactivity (grade 1-3 xGVHD) was observed in 5/10mice in the untransduced group compared to 3/10 in the c-Met IgG4 CARgroup. Tumor formation was not observed at necropsy, and the levels of Tcell engraftment were similar (p=0.39) in the mice engrafted withcontinuous CAR T cells or untransduced primary T cells that werestimulated with anti-CD3 and CD28.

Chimeric Antigen Receptors can Sustain Long Term T-cell Proliferationwithout Transformation

The results presented herein relate to the unexpected finding thatexpression of some CARs containing CD28 and CD3ζ tandem signalingdomains led to constitutive activation and proliferation of primaryhuman T cells. It was observed that some CAR T cells exhibitedconstitutive secretion of large amounts of diverse cytokines andconsequently do not require the addition of exogenous cytokine or feedercells in order to maintain proliferation. This was surprising because inthe numerous previous reports that described CARs endowed with CD28domains (Krause et al, 1998, J Exp Med 188:619-626; Finney et al., 1998,Journal of Immunology 161:2791-2797; Geiger et al., 2001, Blood98:2364-2371; Arakawa et al., 2002, Anticancer Research 4285-4289;Haynes et al., 2002, J Immunol 169(10):5780-6; Maher et al., 2002,Nature Biotechnology 20:70-75; Finney et al, 2004, J Immunol172:104-113; Feldhaus et al., 1997, Gene Ther 4:833-838; Moeller et al.,2004, Cancer Gene Ther 11:371-379; Teng et al., 2004, Hum Gene Ther15:699-708; Friedmann-Morvinski et al., 2005, Blood 105:3087-3093;Westwood et al., 2005, Proc Natl Acad Sci USA 102:19051-19056; Pule etal., 2005, Molecular Therapy 12:933-941; Willemsen et al., 2005, JImmunol 174:7853-7858; Loskog et al., 2006, Leukemia 20:1819-1828;Kowolik et al, 2006, Cancer Res 66:10995-11004; Shibaguchi et al., 2006,Anticancer Res 26:4067-4072; Teng et al., 2006, Human Gene Therapy17:1134-1143; Brentjens et al., 2007, Clin Cancer Res 13:5426-5435;Alvarez-Vallina et al., 1996, Eur J Immunol 26:2304-2309; Gyobu et al.,2004, Cancer Res 64:1490-1495), the proliferation of such tandem CARshas been ligand dependent, and required restimulation of the CAR T cellsin order to maintain proliferation. Here, the results show that onemechanism that can result in the phenotype of CARs with continuous Tcell proliferation is the density of the CAR at the cell surface.

It is believed that this is the first description of “continuous CARs”,i.e. primary T cells that exhibit prolonged exponential expansion inculture that is ligand independent and independent of the addition ofexogenous cytokines or feeder cells. The constitutive secretion of largeamounts of cytokines for several months by non-transformed T cells wasunexpected. The continuous CAR T cells progressively differentiateduring culture towards terminal effector cells and transformation hasnot been observed. The mechanism of the growth phenotype involvescontinuous ligand-independent signal transduction involving canonicalTCR and CD28 signal transduction pathways. One mechanism identified thatleads to continuous CAR T cells is the level of scFv surface expression,as CARs expressed brightly at the cell surface had sustainedproliferation, while CARs expressed at lower levels did not exhibitsustained proliferation and cytokine secretion.

These results are notable for several reasons. The nature of the scFvhas a role in the phenotype, as we have observed the continuous CARphenotype with scFvs that are specific for c-Met and mesothelin but notin the case of FMC63 that is specific for CD 19. An implication of thisfinding is that one cannot assume that the behavior of a signalingdomain coupled to a given scFv will be the same when expressed with adistinct scFv. The method of CAR expression also has an unexpectedcontribution to the growth phenotype. To date, constitutive growth of Tcells when the CARs are expressed by electroporation of mRNA or plasmidsencoding Sleeping Beauty transposons have not observed (Zhao et al.,2010, Cancer Res 70:9062-9072; Huang et al., 2006, Blood 107:483-491;Singh et al., 2008, Cancer Research 68:2961-2971). When expressed usinglentiviral vectors, continuous growth in vectors that employ the EF-1αpromoter have only been observed. In previous studies comparing severalpromoters in lentiviral vectors, it was found that this promoterresulted in more stable and higher level expression in primary CD4 andCD8 T cells (Milone et al., 2009, Mol Ther 17:1453-1464). The particulardesign of the hinge and extracellular domain does not appear to have amajor contribution to the continuous growth phenotype as this phenomenonwith CARs that encode either the longer IgG4 hinge or the shorter CD8αscaffold have been observed. High level expression of the CAR appears tobe necessary for the continuous growth phenotype.

It is believed that this is the first report of constitutive expressionof the endogenous IL-2 gene in primary non-transformed T cells. Previousstudies have shown that constitutive expression of IL-2 and CD25 occursunder conditions that lead to transformation of T cells, mostprominently in HTLV-1 infection (McGuire et al, 1993, J Virol67(3):1590-1599). It is likely that sustained signaling of the CD28cytosolic domain encoded by the CAR is responsible for the constitutivesecretion of IL-2 and numerous other cytokines. It is interesting thatboth HTLV-1 mediated expression of IL-2 by tax and IL-2 secretion drivenby the endogenous CD28 pathway have been reported to be resistant tocyclosporine (Good et al., 1997, J Biol Chem 272(3):1425-1428; June etal., 1987, Mol Cell Biol 7(12):4472-4481), an immunosuppressant thatinhibits the calcineurin phosphatase.

The results presented herein suggest that overexpression of the CD28transmembrane and cytosolic domains in the context of some CARs can leadto constitutive signaling. Thus, it is likely that the regulation ofendogenous CD28 gene expression is a critical determinant of T cellhomeostasis, consistent with studies showing that overexpression of CD28ligands leads to T cell hyperplasia in mice (Yu et al., 2000, J Immunol164:3543-3553).

It is poorly understood why human T cells progressively downregulateCD28 expression with age and cell division (Goronzy et al., 2012, SeminImmunol 24(5):365-72). The constitutive CAR T cells maintained CARexpression at bright levels and had far more rapid downregulation of theendogenous CD28 molecule than classic CARs or non-transduced T cells. Adileucine motif in CD28 contributes to limiting expression of CARs onmouse T cells, and mutating this sequence leads to increased expressionof the CAR (Nguyen et al., 2003, Blood 102(13):4320-5). The constitutiveCAR T cells that have been tested employed the wild type dileucine motifin the CD28 endodomain.

The data presented herein indicates that given a permissive scFv, a5-fold change in the level of expression can lead to the continuous CARphenotype. This may explain why other laboratories have not detectedthis phenomenon using other expression systems.

Previous studies have tested tumor infiltrating lymphocytes (TIL) thatwere transduced to constitutively express IL-2, and the IL-2 TIL did nothave better efficacy than conventional TIL in patients with metastaticmelanoma (Heemskerk et al., 2008, Human Gene Therapy 19:496-510).Similarly, constitutive expression of IL-15 in human CD8 T cells led tothe clonal outgrowth in the absence of exogenous cytokine afterretroviral transduction with the IL-15 gene (Hsu et al., 2007, Blood109:5168-5177).

The safety and clinical benefit with CD19 CARs that use the 4-1BBsignaling domain have been reported (Porter et al., 2011, N Engl J Med365:725-733; Kalos et al., 2011, Sci Transl Med 3:95ra73). T cellsexpressing this CAR have enhanced ligand-independent proliferation(Milone et al., 2009, Mol Ther 17:1453-1464) but do not have the longterm continuous growth phenotype that has been described herein. CARscontaining CD28 signaling domains have now been tested with safety inseveral clinical trials (Savoldo et al., 2011, J Clin Invest121:1822-1825; Brentjens et al., 2011, Blood 118:4817-4828; Kochenderferet al., 2010, Blood 116:4099-4102; Till et al., 2012, Blood119:3940-3950; Kochenderfer et al., 2012, Blood 119:2709-2720). Howeverit is important to note that those trials expressed the CARs aftermanufacturing with a different cell culture system and with a retroviralvector rather than the lentiviral vector that were used in the presentwork. Experiments can be conducted to determine whether continuous CARssuch as those reported here would be useful and safe the clinicalsetting.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An isolated nucleic acid sequence encoding achimeric antigen receptor (CAR), wherein the CAR comprises an anti-c-Metantibody or fragment thereof, an IgG4 hinge domain, a CD28 transmembranedomain, a CD28 costimulatory signaling region, and a CD3 zeta signalingdomain, and further wherein the CAR comprises the amino acid sequence ofSEQ ID NO:
 1. 2. A T cell comprising a nucleic acid sequence thatexpresses a chimeric antigen receptor (CAR), the CAR comprising ananti-c-Met antibody or fragment thereof, an IgG4 hinge domain, a CD28transmembrane domain, a CD28 costimulatory signaling region, and a CD3zeta signaling domain, and further wherein the CAR comprises the aminoacid sequence of SEQ ID NO:
 1. 3. A vector comprising a nucleic acidsequence encoding a chimeric antigen receptor (CAR), the CAR comprisingan anti-c-Met antibody or fragment thereof, an IgG4 hinge domain, a CD28transmembrane domain, a CD28 costimulatory signaling region, and a CD3zeta signaling domain, and further wherein the CAR comprises the aminoacid sequence of SEQ ID NO: 1.